LOCUS       sequence-317655-        5880 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mutated membrane scaffold protein 
            (""ultra-dark"" MSP1D1) for Nanodisc formation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5880)
  AUTHORS   McLean MA, Denisov IG, Grinkova YV, Sligar SG
  TITLE     Dark, Ultra-Dark and Ultra-Bright Nanodiscs for membrane protein 
            investigations.
  JOURNAL   Anal Biochem. 2020 Oct 15;607:113860. doi: 10.1016/j.ab.2020.113860.
            Epub 2020 Aug 1.
  PUBMED    32750355
REFERENCE   2  (bases 1 to 5880)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Anal 
            Biochem."; date: "2020-10-15"; volume: "607:113860. doi"; pages: " 
            10.1016/j.ab.2020.113860. Epub 2020 Aug 1"
COMMENT     Sequence Label: pUD-MSP1D1
FEATURES             Location/Qualifiers
     source          1..5880
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     563..582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(602..679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     885..904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     988..1007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        988..1006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1007..1031
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1046..1068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1081..1101
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             1123..1143
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1732..1749
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1802..1820)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1816..1863
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1900..2355
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1987..2006)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2197..2218
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2447..3262)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2447..3262)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3170..3189
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3254..3273
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3384..3972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3873..3892
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4126..4143
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4158..4297
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4223..4242)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4383..4405
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4399..4590)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5399..5880,1..601))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
       61 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      721 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
      781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
      841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
      901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
      961 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1021 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggt
     1081 catcatcatc atcatcatca cgattatgat attcctacta ctgagaattt gtattttcag
     1141 ggttctacct tcagtaaact tcgcgaacaa ctgggccccg tgacgcagga attcttcgac
     1201 aacctggaaa aagaaaccga gggactgcgt caggaaatgt ccaaagattt agaagaggtg
     1261 aaggccaagg ttcagccatt tctcgatgac tttcagaaaa aattccagga agagatggaa
     1321 ttatttcgtc aaaaggtgga accgctgcgt gcggaactgc aagagggggc acgccaaaaa
     1381 ctccatgagc tccaagagaa gctcagccca ttaggcgaag aaatgcgcga tcgcgcccgt
     1441 gcacatgttg atgcactccg gactcatttg gcgccgtttt cggatgaact tcgccagcgt
     1501 ttggccgcac gtctggaggc gctgaaagaa aacgggggtg cccgcttggc tgagttccac
     1561 gcgaaagcga cagaacacct gagcaccttg agcgaaaaag cgaaaccggc gctggaagat
     1621 ctacgccagg gcttattgcc tgttcttgag agctttaaag tcagttttct gtcagctctg
     1681 gaagaattta ctaaaaagct gaatacccag taagcttgcg gccgactcga gcaccaccac
     1741 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1801 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1861 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1921 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1981 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2041 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2101 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2161 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2221 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2281 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     2341 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2401 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     2461 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     2521 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     2581 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     2641 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     2701 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     2761 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     2821 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     2881 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     2941 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     3001 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     3061 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     3121 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     3181 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     3241 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     3301 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3361 gtagaaaaga tcaaaggatc ttcttgaaat cctttttttc tgcgcgtaat ctgctgcttg
     3421 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3481 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3541 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3601 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3661 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3721 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3781 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3841 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3901 gtcgggtttc gccaccactg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3961 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4021 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4081 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4141 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4201 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     4261 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     4321 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4381 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     4441 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     4501 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     4561 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     4621 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     4681 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     4741 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     4801 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     4861 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     4921 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     4981 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     5041 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     5101 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     5161 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     5221 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     5281 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5341 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5401 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5461 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     5521 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     5581 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     5641 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     5701 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     5761 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     5821 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
//
